Spore photoproduct lyase catalyzes specific repair of the 5R but not the 5S spore photoproduct

Full text of DOI:10.1021/ja807375c

Published on Web 01/29/2009
Spore Photoproduct Lyase Catalyzes Specific Repair of the 5R but Not the 5S
Spore Photoproduct
Tilak Chandra, Sunshine C. Silver, Egidijus Zilinskas, Eric M. Shepard, William E. Broderick, and
Joan B. Broderick*
Department of Chemistry and Biochemistry and the Astrobiology Biogeocatalysis Research Center, Montana State
UniVersity, Bozeman, Montana 59717
Received September 17, 2008; E-mail: jbroderick@chemistry.montana.edu
Scheme 1. Formation and Repair of Spore Photoproduct
Bacterial spores are remarkable in their resistance to chemical
and physical stresses, including exposure to UV radiation. The
unusual UV resistance of bacterial spores is a result of the unique
photochemistry of spore DNA coupled with the efficient repair of
accumulated damage. Exposure of bacterial spores to UV radiation
results in the formation of a methylene-bridged thymine dimer,
5-thyminyl-5,6-dihydrothymine (spore photoproduct, or SP), as the
primary photoproduct.1–3 SP accumulates in UV-irradiated spores;
however, it is rapidly repaired upon germination, thus giving rise
to the extraordinary UV resistance of bacterial spores.4,5 The repair
of SP is catalyzed by the enzyme spore photoproduct lyase (SPL),
and involves the direct reversal of SP to two thymines without base
excision (Scheme 1).
SPL is a member of the radical AdoMet superfamily, and utilizes
a [4Fe-4S] cluster and S-adenosylmethionine (AdoMet) as essential
cofactors in SP repair.6–9 We have previously shown that SP repair
is initiated by abstraction of H · from C6 of SP by an AdoMet-
derived 5′-deoxyadenosyl radical;10,11 this H-atom abstraction is
thought to initiate a radical-mediated ꢀ-scission of the C5-Cbridge
bond in the photoproduct, as originally proposed by Mehl and
Begley.12
While two distinct diastereomers of SP (5R or 5S, Figure 1) could
in principle be formed upon UV irradiation of bacterial spores, only
the 5R configuration is possible for SP formed from adjacent
thymines in double helical DNA, due to the constraints imposed
by the DNA structure.13 The 5S configuration, therefore, is possible
only in less well-defined DNA structures or as an interstrand cross-
link. It was thus quite surprising when two recent reports concluded
that SPL repairs only the 5S, and not the 5R, isomer of a synthetic
SP substrate.14,15 We report here results from HPLC and MS
analysis of in Vitro enzymatic assays on stereochemically defined
synthetic SP substrates demonstrating that SPL specifically repairs
only the 5R isomer of SP. This stereospecific repair of 5R-SP by
SPL is consistent with the longstanding hypothesis that SP is a
result of UV-induced dimerization of adjacent thymines in double-
helical DNA.
open dinucleoside forms of SP, the protected open dinucleosides
(5R and 5S) were converted to closed cyclic phosphotriesters, and
the stereochemical assignments at C-5 were confirmed by NOESY
and ROESY (SI). Stereochemical assignments were based on
initially assigning the pro-S and pro-R hydrogens at C6, with the
pro-R hydrogen appearing downfield of the pro-S hydrogen for
nearly all of the synthetic forms of both diastereomers of SP (SI).
Our assignment of pro-R and pro-S hydrogens differs from that
reported previously, and may account for the disparity between our
results and those previously published.14,15 Proper assignment of
the pro-R and pro-S hydrogens is critical to the determination of
stereochemistry at C5, as the coupling between these protons and
the C5 methyl, as examined by 2D NOESY and ROESY, can then
be used to assign the absolute configuration. Our assignments of
stereochemistry are completely consistent with distances derived
from our computational models of the two diastereomers of SP
(SI).
SPL was cloned from Clostridium acetobutylicum, overexpressed
in Escherichia coli, and purified using a method similar to published
procedures.11 The enzyme contained 2.9 ((0.2) Fe per SPL, and
had UV-visible and EPR spectroscopic properties characteristic
of an iron-sulfur enzyme. The 5R and 5S diastereomers of protected
(N-SEM, O-TES, and O-TBDMS) SP were synthesized using
modifications of published procedures,13,15 and were subsequently
deprotected (Supporting Information (SI)). The structures of the
fully protected, the di-SEM protected, and fully deprotected
1
dinucleoside spore products were confirmed by H and 13C NMR
techniques, and NOESY and ROESY were used to assign the
stereochemistry at C-5 (SI). To remove any potential ambiguity
associated with the assignment of stereochemistry at C-5 in the
Figure 1. Chemical drawings (top) and computational models (bottom)
for the 5R (left) and 5S (right) synthetic spore photoproduct analogues
lacking a DNA backbone.
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10.1021/ja807375c CCC: $40.75
2009 American Chemical Society

C O M M U N I C A T I O N S
constrained to the conformation found in a DNA strand, and also
lacks the phosphodiester bridge and oligonucleotide strand that
likely contribute considerably to substrate binding interactions in
the SPL active site. We have incorporated the protected SP
phosphotriester described in the Supporting Information into an
oligo; however, our initial assays on this indicated that the enzyme
cannot repair the di-SEM protected SP analogue. Carell has also
incorporated a synthetic SP analogue into an oligonucleotide strand,
however the analogue contains no phosphodiester linkage; no assay
data on the resulting oligos were presented.16 We are currently
pursuing deprotection of the SP phosphotriesters (SI) and incor-
poration of them into oligonucleotide DNA, to produce stere-
ochemically defined substrates for further assay and mechanistic
studies.
The results presented herein provide direct and quantitative
evidence for the stereochemical requirements of SP repair by SPL,
demonstrating that SPL repairs specifically the 5R isomer of SP.
The identification of 5R-SP as the substrate of SPL supports the
premise that SP, like the more familiar cyclobutane thymine dimer,
is a result of UV-induced dimerization of adjacent thymines in a
DNA strand. Furthermore, the observation that only 5R-SP is a
substrate for SPL is consistent with the expectation, based on the
constraints imposed by the DNA double helical structure, that 5R
is the SP isomer produced in ViVo upon UV irradiation of bacterial
spore DNA, an expectation supported by a recent report showing
that 5R SP is the diastereomer produced upon UV irradiation of a
TpT dinucleotide.17
Figure 2. HPLC chromatograms showing the time-dependent formation
of thymidine due to repair of R-SP (A) but not S-SP (B), upon incubation
of 1 mM SP with SPL (50 µM), AdoMet (1 mM), DTT (5 mM), and
dithionite (1 mM) in buffer (see SI) at 30 °C. SP elutes at 19.3 (5R) or
20.3 (5S) min and thymidine elutes at 14.4 min under these conditions.
Integration of the thymidine and SP peaks allowed the quantitation of
turnover of each isomer of SP (C).
Acknowledgment. The authors thank the National Institutes of
Health for financial support of this research (GM67804). We
gratefully acknowledge the assistance of Valerie Copie on NMR
experiments and Robert Szilagyi on computational modeling of SP.
Funds to purchase the Bruker 600 MHz NMR were provided by
the NIH (SIG 1-S10RR13878) and the NSF (EPSCOR- Montana).
Assays of SP repair were conducted at 30 °C under anaerobic
conditions; details are provided in SI. Under the HPLC conditions
utilized 5R-SP elutes at 19.3 min, 5S-SP elutes at 20.3 min, and
thymidine elutes at 14.4 min. As can be seen in Figure 2, time-
dependent formation of a peak with the retention time of thymidine
is observed when 5R-SP, but not when 5S-SP, is used as a substrate.
The identity of this emerging peak as thymidine was confirmed by
co-injection with authentic thymidine and by MS analysis of HPLC
fractions corresponding to the peak (SI). The synthetic SP as well
as the SP peaks in Figure 2 (m/z ) 485 (SP) and 507 (SP + Na)),
as well as the peak labeled T in Figure 2 (m/z ) 265 (T + Na)),
gave rise to the expected pattern upon MS analysis. MS analysis
of the small peaks eluting between 15 and 17 min show them to
result from inhomogenous mixture of small peptides, presumably
resulting from protein degradation during the sample workup. These
assays have been performed seven different times, on two different
protein samples, using two different buffers and two different
reducing agents (dithionite or 5-deazariboflavin), and in all cases
SPL repaired the 5R but not the 5S-SP.
Integration of the thymidine and SP peaks in the chromatograms
in Figure 2 have allowed us to quantify the rate of SP repair in this
system (Figure 2C) as ∼0.4 nmol/min/mg of SPL for the R isomer,
and 0 nmol/min/mg of SPL for the S isomer. These rates can be
compared to the rate of 0.33 µmol/min/mg when using B. subtilis
SPL and UV-irradiated plasmid DNA as a substrate.11 Little
additional SP turnover occurs after the 4 hour time point shown in
Figure 2C, likely due to enzyme instability under assay conditions,
and/or to enzyme inactivation. The lower rate of turnover for
synthetic dinucleoside SP relative to SP generated by UV irradiation
of plasmid DNA is not surprising, as the synthetic SP is not
Supporting Information Available: Synthetic methods and NMR
characterization of all synthetic SPs mentioned herein; NOESY and
ROESY of selected compounds; molecular modeling of SPs; mass spec
data on T and SP peaks from the HPLC-based assay. This material is
available free of charge via the Internet at http://pubs.acs.org.
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